AbstractIn this study, twelve campesterol derivatives (2–13) were prepared by esterification reaction at the hydroxy group in C‐3 and catalytic hydrogenation at the carbon‐carbon double bond in C‐5(6). All obtained compounds were characterized by IR, 1H‐NMR, 13C‐NMR, and MS spectra. Campesterol (1) and its derivatives (2–13) were evaluated in vitro against Staphylococcus aureus (ATCC 6538), Streptococcus mutans (ATCC 0046), Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 15442), and Klebsiella pneumoniae (ATCC 10031) using the microdilution method. Among tested compounds, 4, 6, 9, 11, 12, and 13 displayed the best antibacterial activity. Moreover, to support the antibacterial activity experiments, the investigation of molecular interactions of more active compounds, and also compound 1 and neomycin, used as starting material and positive control, respectively, at the binding site of the target proteins was performed using molecular docking simulations. Four compounds (7, 9, 10 and 11) are herein described for the first time.